Super-resolution imaging of histones and DNA reveals that DNA is compacted by groups of nucleosomes -clutches -at the nanoscale level and clutch compaction of DNA is affected by histone tail acetylation especially in highly folded regions containing several nearby clutches. AbstractChromatin organization plays an important role in regulating gene expression. Previously, we showed that chromatin is organized in the form of nucleosome groups or clutches. The size and nucleosome packing density of clutches decreased in hyperacetylated cells having more open chromatin. While hyperacetylation is thought to disrupt histone-DNA and inter-nucleosome interactions, its impact on higher order DNA compaction by groups of nucleosomes in vivo is not clear. To elucidate this question, we carried out two-color super-resolution imaging of histones and DNA in cells treated with the Histone Deacetylase (HDAC) inhibitor Trichostatin A (TSA). We showed that a lower percentage of DNA was associated to clutches in hyperacetylated cells, suggesting a decrease in nucleosome occupancy. We further identified the presence of "clutch" DNA within a nanoscale distance around the clutches. Upon histone hyperacetylation, the radius of the clutch DNA decreased leading to DNA release from the clutches, consistent with disruption of DNA-histone interactions. Finally, the most dramatic decompaction was observed for groups of clutches in close spatial proximity, suggesting that neighboring clutches influence each other´s DNA compaction.